Cyclic AMP (cAMP) is a ubiquitous intracellular second messenger that coordinates diverse cellular functions. It is produced in response to a large variety of extracellular stimuli, including hormones and neurotransmitters. Typically, these agents bind to receptors on the extracellular surface, the receptors activate G-proteins on the intracellular surface and the G-proteins in turn activate adenylyl cyclase, the enzyme that produces cAMP.
cAMP signals are considered to be complex, as evidenced by cAMP's differential regulation of over 200 cellular targets. In addition, the enzymes involved in cAMP metabolism are known to be regulated by numerous other signaling pathways. Unfortunately, as discussed further below, an understanding of cAMP signals has been elusive, due to the fact that current methods for measuring cAMP lack both temporal and spatial resolution. Thus, what is needed are high-resolution means that provided the requisite resolution in order to measure intracellular cAMP.
For example, the standard method for measuring cAMP accumulation within cells is to treat cells with [3H]-adenine to label the ATP pool, and then measure the conversion of [3H]ATP to [3H]cAMP at different time points (See e.g., Evans et al., Mol. Pharmacol., 26:395-404 [1984]). This method is typically done on hundreds of thousands to millions of cells. As a consequence, the method has no spatial resolution and it cannot be used to assess cell-to-cell variability within a population. In addition, it is labor-intensive because cAMP accumulation can be measured only at discrete time points (i.e., in contrast to fluorescence or electrophysiological techniques, it does not provide a continuous readout). This is an important consideration in screening applications. In addition, the method has low temporal resolution, as it is impractical to measure cAMP accumulation in less than 5 second increments. Thus, it is likely that rapid changes in cAMP will be missed when this technique is used.
A second method currently used in the art involves measuring the changes in fluorescence energy transfer between labeled subunits of cAMP-dependent protein kinase, which dissociate upon binding of cAMP. Fluorescent subunits are either prepared biochemically and microinjected (See, Adams et al., Nature 349:694-697 [1991]), or are genetically encoded (See, Zaccolo et al., Nat. Cell. Biol., 2:25-29 [2000]). Although this method does allow for detection of cAMP changes in single cells, it has very low spatial resolution due to limitations in the wavelength utilized (i.e., it is limited by the wavelengths of visible light; 400-800 nm). It also has low temporal resolution, due to the slow reassociation of labeled subunits (i.e., a half-time of 100-200 seconds at typical subunit concentrations; See, Ogreid and Doskeland, Biochem., 22:1686-1696 [1983]), and the tendency of catalytic subunits that catalyze phosphorylation to accumulate in the nucleus (See, Harootunian et al., Mol. Cell. Biol., 4:993-1002 [1993]). In addition, in order to overwhelm endogenous kinase, several micromolar labeled subunits are usually introduced into the test system. This strongly buffers natural cAMP signals, and causes functional alterations of cellular targets due to extensive phosphorylation. Thus, there remains a need in the art for methods and compositions that further elucidate the activities of receptors, G-proteins, phosphodiesterases (PDEs), adenylyl cyclases, and other proteins important in cAMP signalling.